The picornaviruses are the smallest known RNA animal viruses. Translation of the viral genome after infection begins at a single initiation site. Cleavage of the resulting giant polypeptide into functional proteins is accomplished with host and possibly viral-coded proteins. I propose to isolate mutants of encephalomyocarditis (EMC) virus having temperature sensitive defects in the hypothetical viral- coded cleavage enzymes. The following questions are posed: 1) Are enzymes involved in all the cleavage reactions, and, if so, are they viral-coded or host proteins? 2) How many proteolytic enzymes are involved in how many cleavage reactions? To answer these, mutants will be isolated and divided into two groups. One group will contain mutants able to synthesize viral RNA but no infective virions upon HeLa cell infection at the nonpermissive temperature and the oher group will contain mutants unable to synthesize viral RNA at the nonpermissive conditions. With the mutants in the former group, the defect may be in the lack of cleavage to give capsid proteins. Polyacrylamide gel electrophoresis of extracts made from cells infected with these mutants at the nonpermissive temperature should indicate if any capsid precursor polypeptides are accumulating because of defective cleavage. With the mutant group unable to synthesize viral RNA in infected cells at the nonpermissive temperature, the defect may be in the lack of cleavage to give those proteins involved in viral RNA synthesis. With one difference, an experiment similar to the previous one should indicate if any viral polypeptide precursors are accumulating. The difference is that the extracts for electrophoresis are made from infected cells harvested after growth for 3 hrs at the permissive temperature with a subsequent 30 min radioactive labeling period at the nonpermissive temperature. The similar phenotypic effects of temperature sensitive cleavage enzymes and cleavage sites must be distinguished. High mutant reversion rate, leakiness, and pleiotropic effects may also cause difficulties.